Project summary One of the first steps in nervous system development consists in the folding of the neural plate and closure of the neural tube to originate the brain and spinal cord. Failure of neural tube formation leads to neural tube defects (NTDs), which are one of the most common serious birth defects. The causes of NTDs are multiple and both genetic and environmental factors have been identified. Among these factors, the use of antiepileptic drugs during pregnancy increases the incidence of NTDs by unknown mechanisms. In the mature nervous system, antiepileptic drugs decrease excitability by targeting diverse effectors. However, most studies have argued that off-target effects of these drugs are responsible for inducing NTDs in epileptic patients? offspring. Instead, the effect of antiepileptic drugs on embryonic neural excitability remained mostly unexplored because of the prevailing view that neural activity is not apparent at neural plate stages. In contrast, our recently published study demonstrates that glutamate signaling is present in the folding neural plate and is necessary for neural tube formation. Downregulating glutamate signaling directly or by incubating Xenopus laevis embryos with the antiepileptic drug valproic acid causes NTDs. In this study we will discover the molecular mechanisms of neurotransmitter signaling during neural tube formation. Challenging the prevailing view, we hypothesize that vesicular glutamate release from neural plate cells is necessary for neural tube formation. Released glutamate elicits calcium transients in neural plate cells that control expression and function of regulatory neural cell cycle proteins like Sox2, which is a pivotal transcription factor for modulating neural stem cell renewal, proliferation and neurogenesis, depending on its level of expression and posttranslational modifications. By using state-of- the-art imaging and molecular approaches, we will discover the molecular mechanisms and spatiotemporal profile of glutamate release and signaling during neural tube formation. We will identify downstream molecules to glutamate release that control neural plate cell proliferation, their mechanisms of action and impact in the formation of the neural tube. We will examine the role of glutamate signaling on the regulation of Sox2 expression and function during neural plate folding. This study proposes a novel paradigm-shifting model in which neurotransmitter signaling is functional at neural plate stages and is crucial for the formation of the neural tube. The significance of the mechanistic knowledge gained form this study is based on the contribution it will make to the field of NTDs and to the molecular understanding of the regulation of neural stem cell cycle progression, which in turn will be relevant to research on brain tumors, brain and spinal cord injuries, neurogenesis in neurodegenerative and neurodevelopmental disorders. This study, by advancing our mechanistic understanding of neural activity-driven neural tube formation will improve preventative measures for epileptic pregnant women.